The search for alternative methodologies to reduce the number of animals used in biomedical research and toxicology testing has focused on in vitro methods of cultured cells. Our concept is to develop an off-the-shelf frozen microtiter plate ("Cryoplate") which will provide a readily available cell delivery system for in vitro assaying of cytotoxicity. We propose to examine the general hypothesis that cells dispensed in a tissue culture plate and attached to either a natural or synthetic substrate can withstand the rigors of freezing and thawing to provide a cell delivery system for in vitro cytotoxicity assays. Initial studies will utilize cultured bovine corneal cells which permit convenient examination of tolerance to the stresses of cryopreservation, either as individual cells attached directly to the synthetic substrate of the tissue culture plate or as a layer of cells attached to the natural extracellular matrix (ECM) laid down by these cells in culture. It is envisaged that optimization of cryobiological variables will enable development of the Cryoplate concept for use in a wide variety of toxicology and diagnostic laboratories. This product will not only eliminate the need for plating and expanding cells for in vitro testing purposes, but will possibly require handling fewer cells than conventional methods. The basic technology developed in this research may eventually lead to the cryopreservation of cells on other substrates such as miniaturized synthetic chips carrying sensors or other diagnostic tools. PROPOSED COMMERCIAL APPLICATION: It is anticipated that cryopreserved cell delivery systems will be commercialized for chemical testing and diagnostic assays. The methods may be applicable to the development of in vitro cell assay kits for a variety of cell types and purposes. Licensing opportunities will be sought with industry partners for alternative applications.